The invention relates generally to the field of gene delivery into host cells, and more particularly, to gene delivery via viral vectors.
Viral-mediated gene delivery has been described for delivery of therapeutic genes to patients. One limitation of currently known methods is the generation of neutralizing antibodies (NAB) by the patients immune response against viral capsids, which prohibit significant levels of gene expression upon readministration. Thus, methods of gene delivery which circumvent these immune responses are needed.
Covalent modification of proteins and enzymes with functionalized poly(ethylene) glycol (PEG) has been studied. PEG is an uncharged, hydrophilic, linear polymer that is non-immunogenic and has a very low order of toxicity. Recently, O""Riordan et al have developed a process to covalently link various polyethylene glycols to the capsid proteins of adenovirus [O""Riordan et al, Hu Gene Therapy, 10: 1349-1358 (1999)]. However, this method requires incubation for a period of 20 to 24 hours.
What is needed are viral vectors which avoid the limitations of current constructs for re-administration, as well as rapid methods for generating high levels of such constructs.
In one aspect, the invention provides a method for conjugating a recombinant virus with polyethylene glycol to enhance transduction efficiency thereof. This method involves the steps of reacting activated PEG and a recombinant virus at room temperature for about 15 minutes to about 2 hours; and stopping the reaction, thereby obtaining PEG-conjugated virus. Most suitably, the activated PEG and the recombinant virus are reacted at a ratio of about 10:1 polyethylene glycol to virus. Desirably, the recombinant virus is present at a concentration of about 1xc3x971010 to about 1xc3x971015 particles per ml of reaction solution.
In another aspect, the invention provides a PEG-conjugated virus prepared according to the method of the invention.
In yet another aspect, the invention provides a method for increasing transduction efficiency of a recombinant virus which involves delivering a modified recombinant virus according to the invention to host cells.
In still another aspect, the invention provides a method for re-administration of a molecule to a selected host cell via a viral vector. This method involves the steps of contacting the host cell with a PEG-modified virus according to the invention, wherein said virus comprises a molecule for delivery to a host cell; and contacting the host cells with a recombinant virus comprising the molecule.
In yet a further aspect, the invention provides a composition useful for delivery of a selected molecule to host cells. The composition contains a PEG-conjugated virus prepared according to the method of the invention and a physiologically acceptable carrier.
In still a further aspect, the invention provides a composition that enhances the physical stability of viral vectors. This composition contains a recombinant viral vector comprising a molecule for delivery to host cells, sucrose, and mannitol, wherein the ratio of sucrose to mannitol is about 1 to about 1. Desirably, the composition is lyophilized to a final moisture content of about 1.2% to about 1.7%.
Other aspects and advantages of the invention will be apparent from the following detailed description of the invention.